At the time of this invention approximately 50 million people worldwide are infected by HIV, many of which are at a stage of their disease where antiretroviral drug therapy is medically indicated. Disease staging of HIV is done by counting specific immune cells in blood several times per year. These cells progressively decrease in number over a span of many years to a level where immunity is impaired and fatal opportunistic infection occurs. This condition is known medically as AIDS. When cell counting is used to monitor specific immune cells in blood and the results are used to time the administration of antiretroviral therapy, the procedure is 90% successful in establishing remission that lasts for several years. Without this the rate of remission is only approximately 20%. It is now an unquestioned axiom that immune cell counting must guide antiretroviral therapy in treating HIV.
It is estimated that 95% of individuals infected with HIV live in regions where skilled laboratory staff do not exist. These are needed to operate “flow cytometers”, which are the instruments used to count immune cells. Flow cytometers are classified as highly complex systems by most regulatory agencies because they need many skilled manual steps to prepare patient blood samples, and they are mechanically and electronically unstable, requiring careful adjustment during the day. The training time for a flow cytometry operator is measured in years. The rate at which the HIV infection epidemic has grown and established a death rate that is now estimated to be 6,000 per day, has bested all efforts to create sufficient numbers of technicians and flow cytometry centers in the less developed parts of the world.
Automation with internal hardware and software checks, such as that developed for the PointCare AuRICA system (PointCare Technologies, Inc., Marlborough, Mass. USA), has eliminated all of the manual steps needed to prepare samples for flow cytometry analysis. This would have a major impact on the level of training needed for flow cytometer operation if it were not for two, remaining difficult problems. The first is training operators to use so-called external control materials (assayed artificial or surrogate blood samples) to verify that the flow cytometer is actually functioning within specification at the beginning and/or end of each day. The second is that these external control materials require an uninterrupted “cold chain” for shipment and storage. Maintaining a cold chain requires training and a refrigeration infrastructure that is generally missing in the rural developing world. The critical path to develop a flow cytometer that can be used by staff with minimal training in resource-poor areas lies along the elimination of external control materials.
In immune status testing, the primary role of external control materials is to assure that the unstable, temperature sensitive, antibodies that are used as reagents to bind to cell surface antigens and classify cells for counting, have retained their chemical binding capacity. It is not enough to know that the right concentration of reagent antibody is used. In fact, both the concentration and the binding activity must be assured for this key reagent. Antibodies are heat labile proteins that are easily damaged after a few hours in a non-air-conditioned laboratory. When that happens, flow cytometry cell counts will be wrong.
The primary problem with external controls for most of the world is that they are as temperature sensitive as antibodies. They need to be shipped under refrigeration, and rarely last more than one month even under careful re-refrigeration once the container is opened. Reliable refrigeration is not always available and this creates logistical problems for remote clinics and a need for well trained logistics personnel in the clinic. Secondarily, external controls are expensive. Some small clinics need to spend more money to run external controls than to run patient samples. Thirdly, quality is only assured for patient samples run during a specified time after an external control sample is run. It would be advantageous if quality could be assured for every patient sample irrespective of when the sample is run.
The subject of this invention is a method and apparatus that uses the patient's own blood cells during the sample run rather than surrogate, external control materials to check antibody concentration and activity. The blood cells used as an “internal control” are not the ones that decline in numbers during the course of HIV disease progression. The internal controls cells are an immune cell class that carry the same binding sites for antibody as do the immune cell class whose population is affected by HIV. These cells provide a means to control antibody concentration and activity that is independent of the disease and avoids the cost of external control materials. Additionally it provides the advantage of providing a simultaneous, “internal control material” check during every patient sample run; an option that is neither financially feasible nor technically feasible with external control materials.